Circulating Tumor Microemboli Research Articles

Use of circulating tumor cells and microemboli to predict diagnosis and prognosis in diffuse glioma.

Circulating tumor cell (CTC) detection is a promising noninvasive technique that can be used to diagnose cancer, monitor progression, and predict prognosis. In this study, the authors aimed to investigate the clinical utility of CTCs in the management of diffuse glioma. Sixty-three patients with newly diagnosed diffuse glioma were included in this multicenter clinical cohort. The authors used a platform based on isolation by size of epithelial tumor cells (ISET) to detect and analyze CTCs and circulating tumor microemboli (CTMs) in the peripheral blood of patients both before and after surgery. Least absolute shrinkage and selector operation (LASSO) and Cox regression analyses were used to verify whether CTCs and CTMs are independent prognostic factors for diffuse glioma. CTC levels were closely related to the degree of malignancy, WHO grade, and pathological subtypes. Receiver operating characteristic curve analysis revealed that a high CTC level was a predictor for glioblastoma. The results also showed that CTMs originate from the parental tumor rather than from the circulation and are an independent prognostic factor for diffuse glioma. The postoperative CTC level is related to the peripheral immune system and patient survival. Cox regression analysis showed that postoperative CTC levels and CTM status are independent prognostic factors for diffuse glioma, and CTC- and CTM-based survival models had high accuracy in internal validation. The authors revealed a correlation between CTCs and clinical characteristics and demonstrated that CTCs and CTMs are independent predictors for the diagnosis and prognosis of diffuse glioma. Their CTC- and CTM-based survival models can enable clinicians to evaluate patients' response to surgery as well as their outcomes.

The rare circulating tumor microemboli as a biomarker contributes to predicting early colorectal cancer recurrences after medical treatment

Early prognosis of cancer recurrence remains difficult partially due to insufficient and ineffective screening biomarkers or regimes. This study evaluated the rare circulating tumor microemboli (CTM) from liquid biopsy individually and together with circulating tumor cells (CTCs) and serum CEA/CA19-9 in a panel, on early prediction of colorectal cancer (CRC) recurrence. Stained CTCs/CTM were detected by a microfluidic chip-based automatic rare-cell imaging platform. ROC, AUC, Kaplan-Meier survival, and Cox proportional hazard models regarding 4 selected biomarkers were analyzed. The relative risk, odds ratio, predictive accuracy, and positive/negative predictive value of biomarkers individually and in combination, to predict CRC recurrence were assessed and preliminarily validated. The EpCAM+Hochest+CD45− CTCs/CTM could be found in all cancer stages, where more recurrences were observed in late-stage cases. Significant correlations between CTCs/CTM with metastatic stages and clinical treatment were illustrated. CA19-9 and CTM could be seen as independent risk factors in patient survivals, while stratified patients by grouped biomarkers on the Kaplan-Meier analyses presented more significant differences in predicting CRC recurrences. By monitoring the panel of selected biomarkers, disease progressions of 4 CRC patients during follow-up visits after first treatments within 3 years were predicted successfully. This study unveiled the value of rare CTM on clinical studies and a panel of selected biomarkers on predicting CRC recurrences in patients at the early time after medical treatment, in which the CTM and serum CA19-9 could be applied in clinical surveillance and CRC management to improve the accuracy.

CD47 Expression in Circulating Tumor Cells and Circulating Tumor Microemboli from Non-Small Cell Lung Cancer Patients Is a Poor Prognosis Factor.

To isolate, quantify and analyze CTCs/CTMs from metastatic NSCLC patients, identify TGFβRI/CD47 expression in CTCs/CTMs, and correlate with progression-free survival (PFS). Blood (10 mL) was collected at two time-points: T1 (before the beginning of any line of treatment; T2 (60 days after initial collection). CTCs were isolated using ISET®. Immunocytochemistry was conducted to evaluate TGFβRI/CD47 expression. 45 patients were evaluated. CTCs were observed in 82.2% of patients at T1 (median: 1 CTC/mL; range: 0.33-11.33 CTCs/mL) and 94.5% at T2 (median: 1.33 CTC/mL; 0.33-9.67). CTMs were observed in 24.5% of patients and significantly associated with poor PFS (10 months vs. 17 months for those without clusters; p = 0.05) and disease progression (p = 0.017). CTMs CD47+ resulted in poor PFS (p = 0.041). TGFβRI expression in CTCs/CTMs was not associated with PFS. In this study, we observed that CTC/CTM from NSCLC patients express the immune evasion markers TGFβRI/CD47. The presence of CTMs CD47+ is associated with poor PFS. This was the first study to investigate CD47 expression in CTCs/CTM of patients with NSCLC and its association with poor PFS.

Fusion Cell Markers in Circulating Tumor Cells from Patients with High-Grade Ovarian Serous Carcinoma

Cancer is primarily a disease in which late diagnosis is linked to poor prognosis, and unfortunately, detection and management are still challenging. Circulating tumor cells (CTCs) are a potential resource to address this disease. Cell fusion, an event discovered recently in CTCs expressing carcinoma and leukocyte markers, occurs when ≥2 cells become a single entity (hybrid cell) after the merging of their plasma membranes. Cell fusion is still poorly understood despite continuous evaluations in in vitro/in vivo studies. Blood samples from 14 patients with high-grade serous ovarian cancer (A.C. Camargo Cancer Center, São Paulo, Brazil) were collected with the aim to analyze the CTCs/hybrid cells and their correlation to clinical outcome. The EDTA collected blood (6 mL) from patients was used to isolate/identify CTCs/hybrid cells by ISET. We used markers with possible correlation with the phenomenon of cell fusion, such as MC1-R, EpCAM and CD45, as well as CEN8 expression by CISH analysis. Samples were collected at three timepoints: baseline, after one month (first follow-up) and after three months (second follow-up) of treatment with olaparib (total sample = 38). Fourteen patients were included and in baseline and first follow-up all patients showed at least one CTC. We found expression of MC1-R, EpCAM and CD45 in cells (hybrid) in at least one of the collection moments. Membrane staining with CD45 was found in CTCs from the other cohort, from the other center, evaluated by the CellSearch® system. The presence of circulating tumor microemboli (CTM) in the first follow-up was associated with a poor recurrence-free survival (RFS) (5.2 vs. 12.2 months; p = 0.005). The MC1-R expression in CTM in the first and second follow-ups was associated with a shorter RFS (p = 0.005). CEN8 expression in CTCs was also related to shorter RFS (p = 0.035). Our study identified a high prevalence of CTCs in ovarian cancer patients, as well as hybrid cells. Both cell subtypes demonstrate utility in prognosis and in the assessment of response to treatment. In addition, the expression of MC1-R and EpCAM in hybrid cells brings new perspectives as a possible marker for this phenomenon in ovarian cancer.

Detection of circulating tumor cells and evaluation of epithelial-mesenchymal transition patterns of circulating tumor cells in ovarian cancer.

BackgroundCirculating tumor cells (CTCs) have considered to be promising liquid biopsy in cancer due to the intact information of whole cells and the potential to reflect micrometastasis. However, CTCs research are extremely limited in ovarian cancer, probably due to their rarity. The predictive value of CTCs and circulating tumor microemboli (CTM) in metastasis remains to be elucidated in ovarian cancer. This study tried to identify CTCs/CTM in ovarian cancer with considerably positive rate. To preliminarily identify the invasive capacity of CTCs/CTM, the epithelial-mesenchymal transition (EMT) patterns of CTCs/CTM was evaluated. Moreover, for comprehensive understanding of invasiveness of disseminated cells in ovarian cancer, EMT pattern of exfoliated tumor cells in ascites were also confirmed in this study.MethodsPeripheral blood samples and ascites samples were collected from 22 ovarian cancer patients. The Microfiltration combined with morphological analysis was used to detect CTC single cells or cell clusters. Microfiltration combined with morphological analysis was applied in the CTC isolation and identification. EMT was evaluated by immunofluorescence via markers including vimentin and cytokeratin.ResultsMicrofiltration combined with morphological analysis was introduced to detect CTCs/CTM with a positivity rate of 40.9% in ovarian cancer patients. The number of CTC varied from 1 to 8, with CTM number from 4 to 30. CTCs/CTM of all samples have experienced EMT process. Vimentin was expressed in all CTC samples and all tumor cells in ascites, while cytokeratin was expressed in 44.4% (4/9) of CTC samples. There were no significant differences of the clinical parameters between the CTC-positive and CTC-negative patients.ConclusionsThis study showed that both CTCs/CTM and detached tumor cells in ascites might have undergone complete or partial EMT in ovarian cancer. Moreover, microfiltration combined with cytomorphological analysis showed a considerable CTC detection rate.

Cell–Cell Interactions Drive Metastasis of Circulating Tumor Microemboli

Circulating tumor cells are the cellular mediators of distant metastasis in solid malignancies. Their metastatic potential can be augmented by clustering with other tumor cells or nonmalignant cells, forming circulating tumor microemboli (CTM). Cell-cell interactions are key regulators within CTM that convey enhanced metastatic properties, including improved cell survival, immune evasion, and effective extravasation into distant organs. However, the cellular and molecular mechanism of CTM formation, as well as the biology of interactions between tumor cells and immune cells, platelets, and stromal cells in the circulation, remains to be determined. Here, we review the current literature on cell-cell interactions in homotypic and heterotypic CTM and provide perspectives on therapeutic strategies to attenuate CTM-mediated metastasis by targeting cell-cell interactions.

Homophilic ATP1A1 binding induces activin A secretion to promote EMT of tumor cells and myofibroblast activation

Tumor cells with diverse phenotypes and biological behaviors are influenced by stromal cells through secretory factors or direct cell-cell contact. Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia with fibroblasts as the major cell type. In the present study, we observe enrichment of myofibroblasts in a juxta-tumoral position with tumor cells undergoing epithelial-mesenchymal transition (EMT) that facilitates invasion and correlates with a worse clinical prognosis in PDAC patients. Direct cell-cell contacts forming heterocellular aggregates between fibroblasts and tumor cells are detected in primary pancreatic tumors and circulating tumor microemboli (CTM). Mechanistically, ATP1A1 overexpressed in tumor cells binds to and reorganizes ATP1A1 of fibroblasts that induces calcium oscillations, NF-κB activation, and activin A secretion. Silencing ATP1A1 expression or neutralizing activin A secretion suppress tumor invasion and colonization. Taken together, these results elucidate the direct interplay between tumor cells and bound fibroblasts in PDAC progression, thereby providing potential therapeutic opportunities for inhibiting metastasis by interfering with these cell-cell interactions.

EGFR/MET promotes hepatocellular carcinoma metastasis by stabilizing tumor cells and resisting to RTKs inhibitors in circulating tumor microemboli

The receptor tyrosine kinases (RTKs) family is well-recognized as vital targets for the treatment of hepatocarcinoma cancer (HCC) clinically, whereas the survival benefit of target therapy sorafenib is not satisfactory for liver cancer patients due to metastasis. EGFR and MET are two molecules of the RTK family that were related to the survival time of liver cancer patients and resistance to targeted therapy in clinical reports. However, the mechanism and clinical therapeutic value of EGFR/MET in HCC metastasis are still not completely clarified. The study confirmed that EGFR/MET was highly expressed in HCC cells and tissues and the phosphorylation was stable after metastasis. The expression of EGFR/MET was up-regulated in circulating tumor microemboli (CTM) to accelerate IL-8 production and resistance to the lethal effect of leukocytes. Meanwhile, highly expressed EGFR/MET effectively regulated the Ras/MAPK pathway and stabilized suspended HCC cells by facilitating proliferation and inhibiting apoptosis. Moreover, EGFR/MET promoted phosphorylation of hetero-RTKs, which was dependent on high-energy phosphoric acid compounds rather than their direct interactions. In conclusion, highly expressed EGFR/MET could be used in CTM identification and suitable for preventing metastasis of HCC in clinical practice.

Identification of Atypical Circulating Tumor Cells with Prognostic Value in Metastatic Breast Cancer Patients.

Simple SummaryIn this study we have isolated and analyzed atypical cells found in the blood of metastatic breast cancer patients using a micro-filtration technic. This technic, being very easy to implement, was also extremely useful for studying circulating tumors cells’ (CTCs) heterogeneity in cancer patients. We highlighted three subsets of CTCs, with different independent unfavorable prognostic values for progression-free and overall survival. We demonstrated that these cells can further be analyzed by immunofluorescence to narrow their molecular profiles and identify specific characteristics. Moreover, we identified a subset of CTCs, for which positivity might be a useful stratification tool to select patients more susceptible to benefit from early clinical trials testing novel therapeutics, which frequently enroll late-stage, already heavily pre-treated and thus poor-responder patients.Circulating tumor cells have a strong potential as a quasi-non-invasive tool for setting up a precision medicine strategy for cancer patients. Using a second-generation “filtration-based” technology to isolate CTCs, the Screencell™ technology (Sarcelles, France), we performed a large and simultaneous analysis of all atypical circulating tumor cells (aCTCs) isolated from the blood of metastatic breast cancer (mBC) patients. We correlated their presence with clinicopathological and survival data. We included 91 mBC patients from the PERMED-01 study. The median number of aCTCs was 8.3 per mL of blood. Three subsets of aCTCs, absent from controls, were observed in patients: single (s-aCTCs), circulating tumor micro-emboli (CTM), and giant-aCTCs (g-aCTCs). The presence of g-aCTCs was associated with shorter progression free survival and overall survival. This study highlights the heterogeneity of aCTCs in mBC patients both at the cytomorphological and molecular levels. In addition, it suggests the usefulness of the g-aCTC subset as a prognostic factor and a potential stratification tool to treat late-stage mBC patients and improve their chances of benefiting from early clinical trials.

Targeted downregulation of HIF-1α for restraining circulating tumor microemboli mediated metastasis

Tumor metastasis is directly correlated to poor prognosis and high mortality. Circulating tumor cells (CTCs) play a pivotal role in metastatic cascades, of which CTC clusters is highly metastatic compared to single CTCs. Although platelets and neutrophils within the bloodstream could further exacerbate the pro-metastatic effect of single CTCs, the influence of platelets and neutrophils on CTC clusters mediated metastasis remains unclear. In this study, a pro-metastatic complex composed of CTC clusters, platelets and neutrophils, namely circulating tumor microemboli (CTM), was identified in vivo among different metastatic tumor, which was demonstrated with highly upregulation of hypoxia-inducible factor-1α (HIF-1α). While knock-out of HIF-1α or therapeutically downregulating of HIF-1α via HIF-1α inhibitor (BAY87–2243)-loaded neutrophil cyto-pharmaceuticals (PNEs) could efficiently restrain CTM mediated lung metastasis. The underlying mechanism of metastasis inhibition was attributed to the downregulation of HIF-1α-associated PD-L1, which would enhance immune response for inhibiting metastatic cells. Thus, our work here illustrates that hypoxia was an essential factor in promoting CTM colonization in lung. More importantly, we provide a promising strategy by targeted downregulation of HIF-1α in CTM via neutrophil cyto-pharmaceuticals for treatment of CTM mediated metastasis.

Prognostic value of circulating tumor cells and analysis of clinicopathological factors in liver cancer.

The number of circulating tumor cells (CTCs) and the presence of circulating tumor microemboli (CTM) were determined in the peripheral blood of patients with liver cancer (LC). The relationship between CTCs, CTM, clinicopathologic features, and prognosis of LC was analyzed. The objective of this study was to determine the diagnostic and prognostic value of CTCs/CTM in LC. Patients with LC were enrolled between May 2013 and August 2017, and 67 patients were included in the study. Overall survival curves were built using the Kaplan-Meier method and the log-rank test to identify risk factors. The results were analyzed using a Cox proportional hazards model and expressed as hazard ratio and 95% confidence interval (95% CI). CTCs and either CTCs or CTM were detected in 27 patients (40.3%) and 29 patients (43.3%). CTM were found in four patients. One-year, 3-year, and 5-year survival rates were 42%, 20%, and 15%, respectively. Univariate Cox regression analysis showed that alpha-fetoprotein (AFP), number of CTCs, presence of CTM, and positive CTC/CTM were associated with survival time. Multivariate Cox regression analysis showed that alpha fetoprotein (AFP), number of CTCs, and presence of CTM were independent risk factors for survival in patients with LC. There was no significant correlation between the number of CTCs, the presence of CTM, and clinicopathologic factors. AFP, number of CTCs, and presence of CTM were independent risk factors for survival in patients with LC.

Smart Material‐Integrated Systems for Isolation and Profiling of Rare Cancer Cells and Emboli

This review presents a broad aspect of smart material‐integrated systems for isolating and profiling rare circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) to provide physiological, biological, and mechanistic insights into cancer research. In particular, CTCs/CTM have emanated as essential pieces of evidence that can reveal clonal evolution, tumor heterogeneity, and disease progression within the metastatic cascade. Morphologies, cellular compositions, and rarity of CTCs/CTM make them difficult to track and isolate for profiling distinct molecular characteristics in the case of metastatic potential. Accordingly, with the advanced‐characterization techniques, examining the aspects of the specific surface markers of CTCs/CTM, epithelial‐to‐mesenchymal (EMT), mesenchymal‐to‐epithelial (MET) transitions, and timing of tumor cell dissemination would assist us to understand cancer biology and metastatic characteristics. Existing clinical and research methods for the enrichment and isolation of these sporadic cells depend on mainly conventional methods with low‐yield and expensive features. Owing to their specialized functions and analytical performances, smart material‐based technologies hold an enormous impact not only on cell detection, but also on cell isolation for downstream analyses. Herein, the main reasons for cell isolation are discussed and the recent developments in CTCs/CTM approaches for identifying further methods and future perspectives are elaborated on.

A Graphene Oxide-interfaced Microfluidics System for Isolating and Capturing Circulating Tumor Cells and Microemboli.

Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) are rare cell species present in peripheral blood and appear in circulatory system during cancer metastasis. As phenotype of single or aggregated CTCs can be different and may present different levels of potential aggressiveness, detecting and capturing both of them are crucial for preventing recurrence as well as achieving early-stage diagnosis. This research presents a microfluidics system aiming at isolating and highly sensitive capturing of CTCs and CTMs. A serpentine channel and a series of bifurcating micro-channels were use to separate CTCs and CTMs from other blood cells. A graphene oxide interface was patterned on glass slide to facilitate antibodies conjugation via click chemistry for capturing CTCs and CTMs, thus achieving multiplex detection in a high specificity and bio-compatibility manner.

Platelet-Lymphocyte Ratio, Circulating Tumor Cells and Circulating Tumor Microemboli as Predictors of Thrombosis in Patients with Gastric Cancer

Background: Cancer-associated thrombosis (CAT) is a major cause of morbidity and mortality in oncology patients. There are no accurate risk assessment tools to predict venous thromboembolism (VTE). Circulating tumor cells (CTCs), circulating tumor microemboli (CTM), and high platelet-lymphocyte ratio (PLR) may predispose to VTE. Objective: To evaluate correlations of CTCs, CTM, and PLR with VTE and progression-free survival (PFS) in gastric cancer patients. Methods: Patients with gastric cancer were recruited (March 2016 to April 2017). CTCs were assayed by ISET at two timepoints: before neoadjuvant treatment (CTC1) and after surgery/before adjuvant therapy (CTC2) for patients with localized disease, and before first-line chemotherapy (CTC1) and after 6 months (CTC2) for patients with metastases. VTE incidence was determined retrospectively. PFS was estimated by Kaplan-Meier analysis. Results: We studied 93 patients. According to Khorana scores, 63 (67.7%) patients were at intermediate and 30 (32.3%) were at high risk for VTE. VTE incidence was 20.4% and CTM were found in 39.8%. VTE developed in 7/37 (18.9%) CTM-positive and in 11/50 (22%) CTM-negative patients (p=0.93). When PLR >288, VTE occurred in 7/14 patients (p=0.005). PLR also associated with poor PFS (p<0.0001). CTC2 was associated with poor PFS (p<0.0001). CTC2, PLR and VTE were independent prognostic factors for PFS (p=0.005, 0.043, and <0.0001 respectively). Conclusion: PLR is a prognostic indicator for PFS and for VTE in gastric cancer. Neither CTC, nor CTM improved risk stratification for VTE in our population.

Circulating tumor cells in breast cancer: clinical and molecular parallels

Background. Breast cancer (BC) is the most common type of malignant neoplasm among women, with a high rate of metastasis. Early non-invasive diagnosis is required to increase the effectiveness of anticancer therapy.Objective: to determine the content of circulating tumor cells (CTCs) and their derivatives in the peripheral blood using the MDA-231 aptamer, compare the results obtained with the clinical and molecular characteristics of BC.Materials and methods. The study included 22 patients with BC. Detection of CTCs and circulating tumor microemboli was carried out in 3.5 ml of the blood of BC patients with the help of the MDA-231 aptamer which is affine for breast cancer cells, labeled with the fluorescent Cy3 dye. The count of CTCs in the blood samples was performed using fluorescent and laser scanning microscopy.Results and conclusions. The content of CTCs and circulating tumor microemboli in the peripheral blood of patients with BC of various molecular subtypes was analyzed using the MDA-231 aptamer. The relationship between the number of CTCs and the molecular biological subtype was revealed. The obtained results show the possible prognostic value of CTCs use for monitoring effectiveness of anticancer therapy and control of recurrence of BC.

Prognostic impact and potential predictive role of baseline circulating tumor cells in locally advanced head and neck squamous cell carcinoma

ObjectivesThe prognostic impact of circulating tumor cells (CTCs) or circulating tumor microemboli (CTM) in locally advanced head and neck squamous cell carcinoma (LA-HNSCC) is yet to be determined, with conflicting results in previous trials. The role of induction chemotherapy (ICT) in the management of LA-HNSCC is controversial with no predictive biomarkers to guide treatment strategy in this scenario. The aim of this trial is to determine the prognostic impact of CTCs and CTM, their biomarkers expression by immunocytochemistry (ICC), and its potential role as predictors of ICT benefit in LA-HNSCC. Materials and MethodsProspective study, with newly diagnosed stage III/IV non-metastatic LA-HNSCC patients treated with curative intent. Blood samples analyzed for CTCs and CTM before treatment using the ISET method. ResultsA total of 83 patients were included. CTCs counts were an independent prognostic factor for overall survival (OS; HR: 1.17; 95 %CI: 1.05–1.31; p = 0.005) and progression free survival (PFS; HR:1.14; 95 %CI: 1.03–1.26; p = 0.007). Using the Lausen and Schumacher technique, 2.8 CTCs/mL for OS and 3.8 CTCs/mL for PFS were defined as the best cut-offs. CTM were detected in 27.7% of patients, correlating with worse PFS (HR = 2.70; IC95%: 1.30–5.58; p = 0.007). MRP-7 expression in CTM correlated with worse OS (HR = 3.49; 95 %CI: 1.01–12.04; p = 0.047) and PFS (HR = 3.62; 95 %CI: 1.08–12.13; p = 0.037). CTCs counts were predictive of complete response to treatment (OR = 0.74; 95 %CI: 0.58–0.95; p = 0.022) and high counts (cut-off 3.8/mL) and CTM were potential predictors of ICT benefit. ConclusionCTCs/CTM had significant prognostic impact and potential role as predictors of ICT benefit in LA-HNSCC.

Specific study of biological tumor cytology: a narrative review.

Tumor metastasis is a very complex invasion. The concept of circulating tumor cells (CTCs), which describes the tumor cells diffusing into the blood, is being increasingly recognized for its role in cancer metastasis as well as it is considered the key step of tumor blood metastasis. Epithelial-mesenchymal transition (EMT) is the reverse transition from mesenchymal phenotypes to an epithelial one. Tumor cells undergoing this process have stronger metastatic potential. Though the biological characteristics of CTCs after EMT can help explain the unknown phenomena in tumor metastasis, the biomarkers of CTCs after EMT are not accurate. Also the correlation between CTCs after EMT and the prognosis of patients and the dynamic transformation of cell groups during treatment are not clear. Tumor metastasis initiating cells (MIC), which eventually lead to metastasis in CTCs, are types of cancer stem cells, or at least tumor cells with many stem cell characteristics. Further investigation of the maintenance of tumor stem cell characteristics of CTCs is required. The circulating tumor microemboli (CTM) can prevent CTCs from losing their nests, and the presence of host cells facilitates special metastasis and helps tumor cells escape immune surveillance. In this paper, we reviewed research on the biological cytological characteristics of CTCs in recent years, and provided relevant supporting evidence for monitoring tumor recurrence, assessing patient prognosis, evaluating the sensitivity of anti-tumor biological drugs, and selecting an individualized treatment plan.

Profiling heterogenous sizes of circulating tumor microemboli to track therapeutic resistance and prognosis in advanced gastric cancer

Circulating tumor microemboli (CTM) aggregated by ≥ 2 circulating tumor cells (CTCs) are more migratory than single CTCs. Aside from the plasticity in their molecular characteristics, which have been considered tumor migration, CTM also possesses high size heterogeneity. This study, therefore, systematically investigated the heterogeneous sizes of CTM and their involvement in therapeutic resistance in 114 patients with advanced gastric cancer (GC) using a pre-established surface molecule-independent subtraction enrichment (SE)-iFISH strategy. CTM, which was pre-therapeutically detected in 33.3% of GC patients, can further form in another 34.78% of patients following chemo-/targeted therapies. The presence of CTM is relevant to liver metastasis as well as higher CTC levels (≥ 5/6 mL). Further size-based profiling of GC-CTM revealed that CTM with 2 CTCs (CTM2) was the dominant subtype, accounting for 50.0% of all detected GC-CTMs. However, CTM with 3–4 CTCs (CTM3–4) specifically associates with chemo-/targeted therapeutic resistance and inferior prognosis. Patients with ≥ 1 CTM3–4/6 mL have shorter median progression-free survival and median overall survival. Unlike CTM2 and CTM3–4, which are detectable in pre-therapy and post-therapy, larger aggregated CTM≥5 (CTM with ≥ 5 CTCs) was only intra-therapeutically detected in four HER2+ GC patients, of which three experienced liver metastases. Obtained results suggested that the cluster size of GC-CTM should be dynamically profiled beyond pre-therapeutic whole CTM enumeration in terms of chemo-/targeted resistance or metastasis monitoring. GC-CTM3–4 could be a potential indicator of therapeutic resistance, while the dynamic presence of GC-CTM≥5 implies liver metastasis in HER2+ GC patients.

The Emerging Role of Liquid Biopsy in Gastric Cancer.

(1) Background: Liquid biopsy (LB) is a novel diagnostic method with the potential of revolutionizing the prevention, diagnosis, and treatment of several solid tumors. The present paper aims to summarize the current knowledge and explore future possibilities of LB in the management of metastatic gastric cancer. (2) Methods: This narrative review examined the most recent literature on the use of LB-based techniques in metastatic gastric cancer and the current LB-related clinical trial landscape. (3) Results: In gastric cancer, the detection of circulating cancer cells (CTCs) has been recognized to have a prognostic role in all the disease stages. In the setting of localized disease, cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) qualitative and quantitative detection have the potential to inform on the risk of cancer recurrence and metastatic dissemination. In addition, gastric cancer-released exosomes may play an essential part in metastasis formation. In the metastatic setting, the levels of cfDNA show a positive correlation with tumor burden. There is evidence that circulating tumor microemboli (CTM) in the blood of metastatic patients is an independent prognostic factor for shorter overall survival. Gastric cancer-derived exosomal microRNAs or clonal mutations and copy number variations detectable in ctDNA may contribute resistance to chemotherapy or targeted therapies, respectively. There is conflicting and limited data on CTC-based PD-L1 verification and cfDNA-based Epstein–Barr virus detection to predict or monitor immunotherapy responses. (4) Conclusions: Although preliminary studies analyzing LBs in patients with advanced gastric cancer appear promising, more research is required to obtain better insights into the molecular mechanisms underlying resistance to systemic therapies. Moreover, validation and standardization of LB methods are crucial before introducing them in clinical practice. The feasibility of repeatable, minimally invasive sampling opens up the possibility of selecting or dynamically changing therapies based on prognostic risk or predictive biomarkers, such as resistance markers. Research is warranted to exploit a possible transforming area of cancer care.

Detection of Genomically Aberrant Cells within Circulating Tumor Microemboli (CTMs) Isolated from Early-Stage Breast Cancer Patients.

Simple SummaryDistant metastases derive from the shedding and dissemination of single cancer cells (CTCs) or circulating tumor emboli (CTMs) into circulation. Previous studies on CTMs were mainly run in patients with metastatic disease; however, we observed that CTMs are more frequently detected in patients with early-stage breast cancer. Here, we collected single CTMs and their relative primary tumor tissue samples in early-stage patients. By studying genomic aberrations, present in tumors cells and absent in normal cells, we predicted the tumor fraction thanks to a statistical model developed from a calibration curve with breast cancer cell lines. The tumor fraction ranged from 8% to 48% and CTMs contained specific and shared alterations with respect to tissue. Thus, CTMs may derive from different regions of the primary tumor or from occult micrometastases. Moreover, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be further explored in follow-up and mechanistic studies.Circulating tumor microemboli (CTMs) are clusters of cancer cells detached from solid tumors, whose study can reveal mechanisms underlying metastatization. As they frequently comprise unknown fractions of leukocytes, the analysis of copy number alterations (CNAs) is challenging. To address this, we titrated known numbers of leukocytes into cancer cells (MDA-MB-453 and MDA-MB-36, displaying high and low DNA content, respectively) generating tumor fractions from 0–100%. After low-pass sequencing, ichorCNA was identified as the best algorithm to build a linear mixed regression model for tumor fraction (TF) prediction. We then isolated 53 CTMs from blood samples of six early-stage breast cancer patients and predicted the TF of all clusters. We found that all clusters harbor cancer cells between 8 and 48%. Furthermore, by comparing the identified CNAs of CTMs with their matched primary tumors, we noted that only 31–71% of aberrations were shared. Surprisingly, CTM-private alterations were abundant (30–63%), whereas primary tumor-private alterations were rare (4–12%). This either indicates that CTMs are disseminated from further progressed regions of the primary tumor or stem from cancer cells already colonizing distant sites. In both cases, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be explored in follow-up and mechanistic studies.